The mechanisms of successful long-term vein graft adaptation to the arterial environment without excessive intimal thickening are still not completely understood. Several members of the Ephrin-Eph pathway have recently been described as developmentally specified critical determinants of vessel identity, with Ephrin- B2 ligand an embryonic determinant of arteries and Eph-B4 receptor an embryonic determinant of veins. We have recently shown in both humans and rats that diminished Eph-B4 expression and loss of venous identity is associated with intimal thickening during vein graft adaptation, and that VEGF-A is an upstream inhibitor of intimal thickening. In addition, we showed increased amounts of the vasculoprotective protein Nogo-B during both human and rat vein graft adaptation. However, it is currently not established whether plasticity exists in vessel specification genes in adults, whether the Ephrin-Eph pathway plays a mechanistic role during vein graft adaptation to the arterial environment, or whether Nogo-B is linked to Eph-B4 signaling pathways. We present our exciting new data that: 1) diminished Eph-B4 gene expression and immunoreactive protein is associated with intimal thickening in the mouse model of vein graft adaptation; 2) we can manipulate Eph-B4 signaling in vivo to directly test the role of Eph-B4 in vein graft adaptation in mice; 3) Nogo-B inhibits intimal thickening during vein graft adaptation; and 4) Nogo-B protein is increased in vein grafts treated with siRNA directed against VEGF-A, linking the Nogo-B pathway with the VEGF-A-Eph-B4 pathway. We hypothesize that decreased expression of venous specification genes without concomitant expression of arterial specification genes leads to excessive intimal thickening, and ultimately vein graft failure. We will test our hypothesis with the following specific aims: Aim I. To determine whether Eph-B4 signaling mediates intimal thickening during vein graft adaptation. Aim II. To determine whether VEGF-A inhibition of intimal thickening is mediated by regulation of vessel identity and Eph-B4 expression. Aim III. To determine whether the vasculoprotective effects of Nogo-B during vein graft adaptation are mediated by regulation of Eph-B4 function. The studies are innovative in that analyses of vein graft identity have not been previously performed in mice, and plasticity of the Eph-B4 pathway has not been previously demonstrated in adults. At the completion of these studies, we expect to identify a role for Eph-B4 in vein graft adaptation and to determine whether strategies to limit intimal thickening during vein graft adaptation by altering vessel identity will be a novel approach for a human clinical trial. PUBLIC HEALTH RELEVANCE: Significant numbers of surgically placed coronary and peripheral vein grafts continue to fail due to excessive intimal thickening with resultant patient morbidity and mortality. Improving results of long-term myocardial revascularization and limb salvage remain critically dependent on understanding the mechanisms of excessive intimal thickening during vein graft adaptation to the arterial environment. This proposal takes a novel approach, manipulation of vessel identity, to control intimal thickening.